Chromatin NaCl fractions (Kc167 cells)

Drosophila Kc167 Salt Extracted Chromatin (Henikoff project, subgroup)

General Description

Drosophila melanogaster Kc167 embryonic cells were prepared for Chromatin-chip experimentation.  Chromatin was isolated from these cells was digested with micrococcal nuclease and extracted with 600 mM NaCl.  The soluble fraction was used in two-color hybridization experiments with NimbleGen genomic tiling microarrays.  The hybridization signal from this fraction was compared to the signal from the input DNA.  Scaled log2(ratios) of signal intensity are shown.

We applied genome-wide profiling to successive salt-extracted fractions of micrococcal nuclease-treated Drosophila chromatin. Chromatin fractions extracted with 600 mM NaCl after digestion contain predominantly mononucleosomes and represent classical active chromatin. Profiles of these low-salt soluble fractions display phased nucleosomes over transcriptionally active genes that are locally depleted of histone H3.3 and correspond closely to profiles of histone H2Av (H2A.Z) and RNA polymerase II. This correspondence suggests that transcription can result in loss of H3.3+H2Av nucleosomes and generate low-salt soluble nucleosomes. Nearly quantitative recovery of chromatin is obtained with 600 mM NaCl; however, the remaining insoluble chromatin is enriched in actively transcribed regions. Salt-insoluble chromatin likely represents oligonucleosomes that are attached to large protein complexes. Both low-salt extracted and insoluble chromatin are rich in sequences that correspond to epigenetic regulatory elements genome-wide. The presence of active chromatin at both extremes of salt solubility suggests that these salt fractions capture bound and
unbound intermediates in active processes, thus providing a simple, powerful strategy for mapping epigenome dynamics.

Protocols

  1. Sample preparation: NimbleGen Hybridization, NimbleGen Scanning, Strand-Displacement Labeling
  2. Other Protocols: Nuclei Chromatin Extraction, RNAi in cell culture, NimbleGen Scaling, NimbleGen Scaling

Experimental Reagents

  1. Arrays: 070928_Henikoff_Dmel_r52_ChIP

Sample Details

  1. Animals/Lines: Kc167

  1. Growth and isolation: RNAi Salt Extractions, RNAi in cell culture


Release Date: 2008-12-16